Cloning and structural characterization of porcine heart aconitase.
A full-length cDNA encoding porcine heart aconitase was derived from lambda gt10 recombinant clones and by amplification of the 5' end of the mRNA. The 2700-base pair (bp) cDNA contains a 29-bp 5' untranslated region, a 2343-bp coding segment, and a 327-bp 3' untranslated region. The porcine heart enzyme is synthesized as a precursor containing a mitochondrial targeting sequence of 27 amino acid residues which is cleaved to yield a mature enzyme of 754 amino acids, Mr = 82,754, having a blocked amino terminus. The NH2-terminal pyroglutamyl residue of the mature enzyme was identified by fast atom bombardment mass spectrometry and sequence analyses of an NH2-terminal peptide. Mature porcine heart aconitase contains 12 cysteine residues. Cysteines 358, 421, and 424 are ligands to the Fe-S cluster in the inactive [3Fe-4S] (Robbins, A. H., and Stout, C. D. (1989) Proteins 5, 289-312) and active [4Fe-4S] (Robbins, A. H., and Stout, C. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) forms. An alignment of the derived porcine heart sequence with 8 cysteine-containing tryptic peptides from bovine heart aconitase (Plant, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189; Plank, D. W., Kennedy, M. C., Beinert, H., and Howard, J. B. (1989) J. Biol. Chem. 264, 20385-20393) shows that 198 of 202 amino acids are conserved and suggests that the two enzymes are virtually identical.
Zheng
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Andrews
P C
PC
Hermodson
M A
MA
Dixon
J E
JE
Zalkin
H
H
D000154:Aconitate Hydratase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003545:Cysteine; D004272:DNA, Mitochondrial / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism; D005784:Gene Amplification; D015723:Gene Library; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010449:Peptide Mapping; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D013552:Swine
D004272:DNA, Mitochondrial; D004274:DNA, Recombinant; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D000154:Aconitate Hydratase; D003545:Cysteine